The gut microbiota have been associated with colorectal cancer (CRC), but the specific microbes, patterns of overall community metabolism, and interactions with the mucosal immune system underlying this association are not well-understood. A subset referred to as sulfate-reducing bacteria (SRB) metabolize dietary sulfur to produce hydrogen sulfide (H2S), which in turn can drive CRC. Elevated levels of H2S are known to cause epithelial DNA damage, but identification of specific SRBs or metabolic steps that drive DNA damage in the human gut is lacking. In addition, interactions between SRBs and the mucosal immune system and intratumoral immune cells are underexplored and may also drive tumorigenesis. Dietary components that may serve as inputs to this process have been implicated as a risk factor for CRC, specifically Western-style diets rich in animal products but low in vegetables and fruits. In contrast, prudent diets, high in vegetables, fruits and whole grains, are associated with lower risk. Western diets have recently been associated with the growth of SRB and the production of H2S, whereas prudent diets may inhibit growth of SRB and are a rich source of glucosinolates, sulfur-containing compounds which are metabolized by gut microbial myrosinase into isothiocyanates with cancer preventive properties. However, it remains unclear how, mechanistically, diet influences SRB abundance and their production of H2S, or how SRB mediate the effect of specific dietary components on CRC risk. Thus, studies are needed to examine the interplay between diet, SRB, and CRC. Our central hypothesis is that Western-style diets, in contrast with prudent diets, promote SRB abundance and function, which in turn influence host-microbe regulation of colonic sulfate metabolism. Specifically, this may perturb the intestinal epithelium and intratumoral immune responses, and promote CRC. To address this hypothesis, we initiated one of the first scalable protocols for assessing the gut microbiota in well-characterized cohorts, including the Health Professionals Follow-up Study and Nurses' Health Study II, with diet and lifestyle data collected prospectively over nearly three decades. Within a subset of these cohorts, we have completed collection of stool specimens sampled concurrently with completion of short-term dietary records at multiple timepoints to complement validated long-term dietary data. We will apply a cutting edge microbial profiling and computational analysis pipeline to these longitudinal samples, offering an unprecedented opportunity to examine dietary factors associated with a gut microbiome enriched with SRB. We will complement these studies with dietary and microbial interventions in bona fide mouse models to bridge correlation to causation and elucidate mechanism. Our transdisciplinary team, including leaders in the Human Microbiome Project, has sustained a long record of collaboration to yield transformative results, leading to novel prevention strategies based upon modification of diet or microbial communities. Defining a mechanistic role for SRB and H2S on carcinogenesis may also illuminate new molecular targets for cancer prevention and therapy.